The COMBIMATRIX CUSTOMARRAY® microarray and ElectraSense microarray are complementary metal oxide semiconductor (CMOS) chips with 12,544 electrodes that can be addressed individually or in user-defined groups. These arrays are available commercially as custom DNA chips with different nucleic acid probe sequences produced at each electrode using sequential electrochemical reactions to add phosphoramidites (Maurer K, Cooper J, Caraballo M, Crye J, Suciu D, et al. (2006) Electrochemically generated acid and its containment to 100 micron reaction areas for the production of DNA microarrays. PLoS ONE 1). Hybridization to probes can be detected using cyanine (Cy) dyes and fluorescent scanners or, alternatively, using horseradish peroxidase (HRP) and enzyme-enhanced electrochemical detection (ECD) on CombiMatrix's microarray readers.
In a paper, a method was first described for fixing capture antibodies (Abs) on the 1000-electrode CustomArray microarray, a predecessor of the current ElectraSense microarray (Dill K, Montgomery D D, Wang W, Tsai J C (2001) Antigen detection using microelectrode array microchips. Analytica Chimica Acta 444: 69-78.) Disclosed in this paper is a synthesis of different DNA probes on individual electrodes and use of Abs tagged with complementary oligonucleotides to self-assemble specifically on individual electrodes of the multiplex array. The array had capture Abs against ricin, Bacillus globigii spores, M13 phage, .alpha.1 acid glycoprotein, and fluorescein. Initially, antigen (Ag) binding was measured optically, using fluorophore-labeled target or reporter Ab. However, in later studies, amperometry was used along with HRP, peroxide, and ortho-phenylenediamine (Dill K, Montgomery D D, Ghindilis A L, Schwarzkopf K R (2004) Immunoassays and sequence-specific DNA detection on a microchip using enzyme amplified electrochemical detection. Journal of biochemical and biophysical methods 59: 181-187; Dill K, Montgomery D D, Ghindilis A L, Schwarzkopf K R, Ragsdale S R, et al. (2004) Immunoassays based on electrochemical detection using microelectrode arrays. Biosensors & Bioelectronics 20: 736-742.) These studies reported that the multiplex microarray and assay demonstrated high specificity and sensitivity in the low pg/ml range. However, a problem with current immunoassays is that the conjugated Abs are fragile, expensive, and difficult to produce reliably. Thus, there is a need in the art to provide an immunoassay that uses more robust, less expensive, and easier to produce Abs. Studies have used a constant voltage with a two compartment electrochemical cell where a reference electrode can maintain the applied voltage. In one study, the authors reported that using a potential pulse technique with a range of 0.6 to 1.2 V versus Ag/AgCl for initial structuring of the Ppy was most suitable for entrapping biologically active materials (Ramanavicius A, Ramanaviciene A, Malinauskas A (2006) Electrochemical sensors based on conducting polyer-pyrrole. Electrochimica Acta 51: 6027-6037).
A study reviewed the physical, electrical and chemical parameters that influence the electropolymerization of pyrrole and identified monomer substitution, electrolyte (dopant), solvent, pH, electrochemical method, and temperature as influencing the formation and characteristics of a Ppy film (Sadki S, Schottland P, Brodie N, Sabouraud G (2000) The mechanisms of pyrrole electropolymerization. Chemical Society Review 29: 283-293).
Ppy belongs to a family of conducting polymers that includes polythiophene and polyaniline, each of which have been used to fix proteins and other biomolecules to electrodes for detection using different electrochemical methods. (Cosnier S (1999) Biomolecule immobilization on electrode surfaces by entrapment or attachment to electrochemically polymerized films. A review. Biosensors & Bioelectronics 14: 443-456; Zhang S, Wright G, Yang Y (2000) Materials and techniques for electrochemical biosensor design and construction. Biosensors & Bioelectronics 15: 273-282; Palmisano F, Zambonin P G, Centoze D (2000) Amperometric biosensors based on electrosynthesised polymeric films. Fresenius Journal of Analytical Chemistry 366: 586-601; Ramanaviciene A, Ramanavicius A (2002) Application of polypyrrole for the creation of immuno sensors. Critical Reviews in Analytical Chemistry 32: 245-252; Vidal J-C, Garcia-Ruiz E, Castillo J-R (2003) Recent Advances in electropolymerized conducting polymers in amperometric biosensors. Microchimica Acta 143; Trojanowicz M (2003) Application of conducting polymers in chemical analysis. Microchimica Acta 143: 75-91; Sadik O A, Ngundi M, Wanekaya A (2003) Chemical biological sensors based on advances in conducting electroactive polymers. Microchimica Acta 143: 187-194; Vestergaard Md, Kerman K, Tamiya E (2007) An overview of label-free electrochemical protein sensors. Sensors 7: 3442-3458; Rahman M A, Kumar P, Park D-S, Shim Y-B (2008) Electrochemical sensors based on organic conjugated polymers. Sensors 8: 118-141; Bakker E (2004) Electrochemical sensors. Anal Chem 76: 3285-3298; Diaz-Gonzales M, Gonzalez-Garcia M B, Costa-Garcia A (2005) Recent advances in electrochemical enzyme immunoassays. Electroanalysis 17: 1901-1918; Ramanavicius A, Ramanaviciene A, Malinauskas A (2006) Electrochemical sensors based on conducting polyer-pyrrole. Electrochimica Acta 51: 6027-6037.) In one study, Ppy was identified for its biocompatibility, its ability to transduce energy into electrical signals, its protective properties against electrode fouling, and its potential for in situ modification (Ramanaviciene A, Ramanavicius A (2002) Application of polypyrrole for the creation of immunosensors. Critical Reviews in Analytical Chemistry 32: 245-252.)
The CombiMatrix microarray with 12,544 microelectrodes supports in situ electrochemical synthesis of user-defined DNA probes. CombiMatrix microarrays were initially developed as highly multiplexed platforms for electrochemistry. The original complementary metal oxide (CMOS) microarray had 1,000 platinum (Pt) electrodes (1K microarray), and it was used to develop the in situ electrochemical synthesis of different DNA probes on individual electrodes (Maurer, K.; Cooper, J.; Caraballo, M.; Crye, J.; Suciu, D.; Ghindilis, A.; Leonetti, J. A.; Wang, W.; Rossi, F. M.; Stover, A. G.; Larson, C.; Gao, H.; Dill, K.; McShea, A. Electrochemically generated acid and its containment to 100 micron reaction areas for the production of DNA microarrays. PLoS One 2006, 1, 34). Hybridization to these probes was detected using enzyme-enhanced electrochemical detection (ECD) (Dill, K.; Montgomery, D. D.; Ghindilis, A. L.; Schwarzkopf, K. R. Immunoassays and sequence-specific DNA detection on a microchip using enzyme amplified electrochemical detection. J Biochem. Biophys. Methods 2004, 59, 181-187). The second generation microarray with 12,544 electrodes was mounted in a ceramic slide that was designed so that the chip could be read on a commercial fluorescent microarray reader. The 12K CUSTOMARRAY® microarray is commercially available as a custom gene chip that has been used for a variety of genomic assays (e.g., genotyping, gene expression, SNP analysis, etc.). CombiMatrix also developed the ELECTRASENSE® microarray and microarray reader based on ECD. In comparative studies, ECD provides comparable results to fluorescence detection (Roth, K. M.; Peyvan, K.; Schwarzkopf, K. R.; Ghindilis, A. Electrochemical detection of short dna oligomer hybridization using the combimatrix electrasense microarray reader. Electroanalysis 2006, 18, 1982-1988; Ghindilis, A. L.; Smith, M. W.; Schwarzkopf, K. R.; Roth, K. M.; Peyvan, K.; Munro, S. B.; Lodes, M. J.; Stover, A. G.; Bernards, K.; Dill, K.; McShea, A. CombiMatrix oligonucleotide arrays: genotyping and gene expression assays employing electrochemical detection. Biosens. Bioelectron. 2007, 22, 1853-1860). The latest version of the ElectraSense microarray reader is a palm-sized instrument that interfaces with a personal computer through a USB connection, which provides a data link and power to the reader.
The microarray offers unique capabilities for applications where the electrochemical synthesis or deposition of different molecules on electrodes and different methods of detection are required. The 1K microarray was used to synthesize coumarin or to demonstrate a site-selective hetero-Michael reaction on individual electrodes (Tesfu, E.; Roth, K.; Maurer, K.; Moeller, K. D. Building addressable libraries: Site selective coumarin synthesis and the “real-time” signaling of antibody-coumarin binding. Org. Lett. 2006, 8, 709-712; Stuart, M.; Maurer, K.; Moeller, K. D. Moving known libraries to an addressable array: A site-selective hetero-Michael reaction. Bioconjug. Chem. 2008, 19, 1514-1517). Successful execution of these chemistries was determined using fluorescence detection and cyclic voltammetry (CV). The array has been used with fluorescence detection and time-of-flight secondary ion mass spectrometry to demonstrated molecular synthesis using Wacker oxidations (Chen, C.; Nagy, G.; Walker, A. V.; Maurer, K.; McShea, A.; Moeller, K. D. Building addressable libraries: The use of a mass spectrometry cleavable linker for monitoring reactions on a microelectrode array. J. Am. Chem. Soc. 2006, 128, 16020-16021).
Immobilizing DNA to electrode surfaces using Ppy was originally reported by Minehan et al. (Minehan, D. S.; Marx, K. A.; Tripathy, S. K. Kinetics of DNA binding to electrically conducting polypyrrole films. Macromolecules 1994, 27, 777-783). Since that finding, numerous studies have been done using this and other electroactive polymers as described in recent reviews (Bakker, E. Electrochemical sensors. Anal. Chem. 2004, 76, 3285-3298; Daniels, J. S.; Pourmand, N. Label-free impedance bio sensors: opportunities and challenges. Electroanalysis 2007, 19, 1239-1257; Rahman, M.; Kumar, P.; Park, D. S.; Shim, Y. B. Electrochemical sensors based on organic conjugated polymers. Sensors 2008, 8, 118-141; Peng, H.; Zhang, L.; Soeller, C.; Travas-Sejdic, J. Conducting polymers for electrochemical DNA sensing. Biomaterials 2009, 30, 2132-2148; Galandoava, J.; Labuda, J. Polymer interfaces used in electrochemical DNA-based biosensors. Chem. Pap. 2009, 63, 1-14; Batchelor-McAuley, C.; Wildgoose, G. G.; Compton, R. G. The physicochemical aspects of DNA sensing using electrochemical methods. Biosens. Bioelectron. 2009, 24, 3183-3190; Park, J. Y.; Park, S. M. DNA Hybridization sensors based on electrochemical impedance spectroscopy as a detection tool. Sensors 2009, 9, 9513-9532.)
Most of the studies reported on using label less detection (e.g., CV and electrochemical impedance spectroscopy) for measuring DNA hybridization. More relevant to our findings are those reported by investigators at CIS Bio international and CEA (Livache, T.; Roget, A.; Dejean, E.; Barthet, C.; Bidan, G.; Teoule, R. Preparation of a DNA matrix via an electrochemically directed copolymerization of pyrrole and oligonucleotides bearing a pyrrole group. Nucleic. Acid. Res. 1994, 22, 2915-2921; Livache, T.; Fouque, B.; Roget, A.; Marchand, J.; Bidan, G.; Teoule, R.; Mathis, G. Polypyrrole DNA chip on a silicon device: example of hepatitis C virus genotyping. Anal. Biochem. 1998, 255, 188-194; Caillat, P.; David, D.; Belleville, M.; Clerc, F.; Massit, C.; Revol-Cavalier, F.; Peltie, P.; Livache, T.; Bidan, G.; Roget, A.; Crapez, E. Biochips on CMOS: An active matrix address array for DNA analysis. Sens. Actuat. B: Chem. 1999, 61, 154-162; Cuzin, M. DNA chips: A new tool for genetic analysis and diagnostics. Transfus. Clin. Biol. 2001, 8, 291-296; Livache, T.; Maillart, E.; Lassalle, N.; Mailley, P.; Corso, B.; Guedon, P.; Roget, A.; Levy, Y. Polypyrrole based DNA hybridization assays: study of label free detection processes versus fluorescence on microchips. J. Pharm. Biomed. Anal 2003, 32, 687-696.) This group developed a CMOS microarray with 128 addressable electrodes, and they co-polymerized pyrrole with pyrrole-conjugated DNA probes to create a multiplexed gene chip for the fluorescence detection of hybridization.
A number of investigators have relied on entrapment to immobilize unmodified DNA to Ppy; however, more have modified the DNA, the Ppy, or both to create a covalent attachment between one end of the DNA (usually the 5′-end) and the Ppy. This provides a secure and oriented fixation of the DNA to the Ppy that is often illustrated as a lawn of vertical strands standing perpendicular to the Ppy (Peng, H.; Zhang, L.; Soeller, C.; Travas-Sejdic, J. Conducting polymers for electrochemical DNA sensing. Biomaterials 2009, 30, 2132-2148.)
Minehan et al. and Gambhir et al. reported that the binding of DNA to Ppy is consistent with electrostatic adsorption between the fixed negatively charged phosphates forming the backbone of the DNA and the mobile positively charged defect structures of the Ppy, which favor hydrogen bonding between the phosphates and Ppy ring nitrogen atoms (Minehan, D. S.; Marx, K. A.; Tripathy, S. K. DNA binding to electropolymerized polypyrrole: The dependence on film characteristics. J. Macromol. Sci. Part A: Pure Appl. Chem. 2001, 38, 1245-1258; Gambhir, A.; Gerard, M.; Jain, S. K.; Malhotra, B. D. Characterization of DNA immobilized on electrochemically prepared conducting polypyrrole-polyvinyl sulfonate films. Appl. Biochem. Biotechnol. 2001, 96, 303-309). However, De Giglio et al. demonstrated that cysteine binds to Ppy electropolymerized on platinum or titanium electrodes (De Giglio, E.; Sabbatini, L.; Zambonin, P. G. Development and analytical characterization of cysteine-grafted polypyrrole films electrosynthesized on Pt- and Ti-substrates as precursors of bioactive interfaces. J. Biomater. Sci. Polym. Ed. 1999, 10, 845-858). They presented evidence from X-ray photoelectron spectroscopy that cysteine forms a covalent bond through its sulfur atom by nucleophilic attack on the positive sites of the pyrrole ring. More recently, Zhou et al. reported on immobilizing 5′cys-terminated DNA probes to electropolymerized polyaniline via a nucleophilic substitution reaction and measuring hybridization using CV (Zhou, Y.; Yu, B.; Guiseppi-Elie, A.; Sergeyev, V.; Levon, K. Potentiometric monitoring DNA hybridization. Biosens. Bioelectron. 2009, 24, 3275-3280).
Ramanvicius et al. used Ppy fluorescence quenching to develop an immunoassay against bovine leukemia virus protein gp51 (Ramanavicius, A.; Kurilcik, N.; Jursenas, S.; Finkelsteinas, A.; Ramanaviciene, A. Conducting polymer based fluorescence quenching as a new approach to increase the selectivity of immunosensors. Biosen. Bioelectron. 2007, 23, 499-505). They attributed the quenching to the proximity of the Cy5 to the delocalized .pi.-.pi. electrons in the Ppy backbone, as described by Song et al. (Song, X.; Wang, H. L.; Shi, J.; Park, J. W.; Swanson, B. I. Conjugated polymers as efficient fluorescence quenchers and their applications for bioassays. Chem. Mater. 2002, 14, 2342-2347). Livache et al. did not describe fluorescence quenching by Ppy in their development of a DNA chip that used phycoerythrin as the fluorescent marker; however, they did note that fluorescence increased with increasing Ppy thickness and with a T-linker of increasing length between the pyrrole and the oligonucleotide 5′ end (Livache, T.; Fouque, B.; Roget, A.; Marchand, J.; Bidan, G.; Teoule, R.; Mathis, G. Polypyrrole DNA chip on a silicon device: example of hepatitis C virus genotyping. Anal. Biochem. 1998, 255, 188-194). The Ppy thickness used by these investigators was 20 nm, which was produced by dipping the electrode in 20 mM pyrrole with 1 μM pyrrole-conjugate oligonucleotide and electro-copolymerizing them using CV until a charge of 250 nC was reached.
Neoh et al. [29] and Ando et al. [30] reported that elevated temperatures (100-200° C.) reduced the conductivity of Ppy through a number of possible mechanisms.